Anticoagulants are substances that dampen, reduce or abolish the ability of blood, or blood plasma, to coagulate—anticoagulants reduce the rate by which the fibrinogen is converted into fibrin.
Some anticoagulants are naturally occurring, endogenous, and play important physiological roles in limiting the onset, the speed of propagation and the spatial extension of blood coagulation in vivo. Other anticoagulants appear under pathological conditions. There are anticoagulants found in snake-toxins and in excretions of mosquitoes, leaches, bats and ticks. A growing group of anticoagulants are man-made substances created for the purposes of treating and preventing thromboembolic disorders.
Blood coagulation is a complex phenomenon involving a multitude of interacting molecules found in blood plasma or on the surface of engaged cells. There are numerous cellular and molecular interactions, including many enzymatically catalyzed reactions, which may be down-regulated or disrupted, by the action of anticoagulants.
The direct acting anticoagulants inhibit, hamper or abolish the enzymatic activity of one or both of the critical enzymes in the coagulation process, thrombin (FIIa) or activated coagulation factor X (FXa). Indirect acting anticoagulants work in some other way, one of which is to enhance the potency of direct acting inhibitors. Of anticoagulants in general therapeutic use, heparin is an indirect acting anticoagulant and the vitamin-K antagonists (warfarin) are general reducers of coagulation factor levels. Direct acting anticoagulants are hirudin (a protein of the leach) and the recently created inhibitors of FIIa or FXa. It is these new direct acting anticoagulants, particularly those that can be administered orally, DOACs or as also called NOACs (Non-Vitamin K Oral Anticoagulants), that are causing a profound change within clinical, and laboratory medicine.
The fact that anticoagulants have different modes of action poses a challenge to laboratory medicine in devising methods by which the anticoagulants can be determined. To a degree, this challenge is exasperated by recent pharmacological successes in creating new substances to treat thromboembolic disorders, see e.g. J. Harenburg, S. Marx and R. Kramer, Determination of the anticoagulation effects of the new oral anticoagulants: an unmet need, Expert Review of Haematology, February 2012, Vol. 5, No. 1, Pages 107-113.
At central hospital laboratories, where there is a variety of tests available and qualified personnel to interpret the results, detection and determination of the DOAC appears relatively straightforward—the FIIa inhibitors and FXa inhibitors may be assayed by thrombin time (TT) tests or the FXa tests, respectively. If standard variants of these tests do not fulfill the needs, some modification or “dilution” of the standard variants will. At primary care centers, and at places, sites, within hospitals but remote from the central laboratory, “off-site”, the situation is different. Characteristic for laboratory medicine at POC-sites (Point of Care) is that the number of different laboratory tests is limited because the physical space of such laboratory sites is limited and because the availability of qualified laboratory personnel is limited. Within coagulation the most available POC test is the prothrombin test (PT-test) with the results expressed in INR (international normalized ratio, a ratio between the PT of the sample and a normal PT normalized by being powered to normalizing exponent, a sensitivity index). Other “routine” or “common” coagulation test are activated partial thromboplastin time (APTT), activated coagulation time (ACT) and the mentioned TT. It is the hope of visionary experts in the coagulation field that these “routine” or “common” coagulation tests can be adapted, modified, “diluted”, to fulfill the POC need in assaying the “new” anticoagulants, particularly the NOAC. Such thoughts/hopes are for example expressed by E. J. Favaloro and G. Lippi, The new oral anticoagulants and the future of haemostasis laboratory testing, Biochemia Medica 2012; 22(3):329-41.
Because PT-INR is the most commonly available POC-test for coagulation measurements, a modified PT-test or “diluted PT” by which the NOAC can be determined is highly desirable. There is reported work in such direction. The low sensitivity of most PT-tests for direct FXa inhibitors, such as rivaroxaban, is known. C. Kluft discloses in EP 2 405 274 A1 that PT-tests that are affected by a certain snake venom, RVV-V, also show sensitivity toward rivaroxaban, and discloses the use of a PT-test that employ such thromboplastins.
The hopes of finding a way to determine NOAC by some PT-test, modified or not, has during the recent years declined. In January of 2014, T. Lindahl, a member of expert group in coagulation of the external quality organization of Sweden, EQUALIS, reported on studies performed by the expert group on one FXa-inhibiting substance aprixaban. The conclusion was that none of the many commercially available PT or APTT tests was of use in determining aprixaban at clinically relevant concentrations in blood plasma. FXa-tests, on the other hand worked well for this purpose.
Efforts to correct PT-results for the variable anticoagulant effect of non-functioning coagulation factors, pivka, found in the blood of patients on treatments with vitamin-K antagonists, warrant to be mentioned. U.S. Pat. No. 7,767,459 B2 (J. Horsti) provides for this by measuring PT of blood plasma by the standard protocol of any given PT method, and also measuring the same after a pre-dilution of the plasma with a physiological buffer such as 9 g/l NaCl. The PT results, expressed either in seconds or in INR, are then plotted against the degree of final plasma dilution and extrapolated to zero. The PT of the plasma at final dilution of zero, reduced by the same for a normal plasma, is taken as a measure of the pivka-effect and is used to correct the original PT-result.
New coagulation tests have been devised with the aim to determine several different kinds of anticoagulants, particularly heparins and NOACs.
These efforts demonstrate the clinical importance of determining these anticoagulants.
See Calatzis A, Peletz D, Haas S, Spannagl M, Rudin K, Wilmer M, Prothromibnase-induced Clotting Time Assay for Determination of the Anticoagulant Effects of UFH and LMWH, Fondaparinux, and Thrombin Inhibitors, Am J Clin Pathol 2008; 130: 446-454, and Samama M M, Martinoli J L, LeFlem L, Guinet C, Plu-Bureau G, Depasse F, Assessment of laboratory assays to measure rivaroxaban—an oral, direct factor Xa inhibitor, Thromb Haemost 2010; 103/4: 815-825.
Relevant background to the present invention is the distinction between wet-chemistry methods and dry chemistry methods. Wet-chemistry is defined by the mixing of a volume of the sample, in a coagulation assay of blood or blood plasma, and a volume of reagent. The sample is thus diluted to a certain degree in the reaction mixture. In dry-chemistry this dilution does not occur. The sample is mixed, or contacted, with reagent substance in a dry form and there is no dilution of the sample. Most POC-tests are dry-chemistry tests because they can often be presented in an easy to use format, e.g. a strip or a chip. The operator needs typically only to add a small volume of the sample. A disadvantage of dry-chemistry methods is that the tests are more difficult to modify. One dimension of the freedom granted by the wet-chemistry procedures, variation of the degree of sample dilution, is not available with dry-chemistry. Every mention of a “diluted” modification of a “routine” test has wet-chemistry as a prerequisite.